As monoclonal antibodies that recognize the CD20 antigen, B1, 2B8 (chimeric antibody name is rituximab), 1F5, 2H7 and so forth are known. Above all, rituximab, a chimeric anti-CD20 monoclonal antibody developed by IDEC Pharmaceuticals Corporation, U.S., has been established as a standard therapeutic agent for low malignancy non-Hodgkin's lymphoma (NHL), and found to have a therapeutic effect on many B cell-mediated immunological diseases. For example, it is said to be effective for, in addition to malignant tumors such as chronic lymphatic leukemia, autoimmune diseases in which a pathogenic autoantibody appears to be involved such as autoimmune hemolytic anemia and idiopathic thrombocytopenia purpura, and inflammatory diseases such as chronic rheumatoid arthritis and multiple sclerosis (Non-patent documents 14 to 17).
CD20 is a molecule present on the B lymph cell surface and expression thereof is seen in normal B cells in peripheral blood, spleen, tonsil and bone marrow and so forth as well as B cells in most of malignant tumors. This molecule comprises 297 amino acid residues, penetrates a cell membrane four times, and has both the C-terminus and N-terminus inside the cell, and has the only extracellularly exposed loop with no sugar chain consisting of 43 amino acid residues between the third and fourth transmembrane domains (Non-patent documents 1 and 9). The CD20 molecule is thought to usually exist as a tetramer, and further form a heterocomplex with other minor components (Non-patent document 18). Since the CD20 protein is not secreted out of the cell or cleaved, and in addition, it is hardly taken up into the cell by antibody binding, it can be expected that a cytotoxic mechanism based on an antibody directed to it against a target cell effectively works (Non-patent documents 1 to 3).
In spite of the small molecular size thereof, CD20 shows diversity of epitope partly due to the effect of the expression form thereof as a complex outside the cell, and antibodies binding to it mediate variously different biological responses. For example, activities such as down-regulation of B cell receptors, increase of expressions of MHC class II antigens and adhesion molecules, activation of Ca2+ release in the presence of hyper-cross-linking, inhibition of lymphocyte function-associated antigen 1 non-dependent homotypic adhesion, induction of apoptosis and the opposite activity, promotion of cell growth, vary significantly (Non-patent documents 4 to 13). The typical examples of anti-CD20 antibody, rituximab, B1, 1F5 and 2H7, also have different characteristics and biological functions, and a reference to a “monoclonal antibody binding to CD20” alone cannot specify the biological properties thereof.
The molecule that constitutes the extracellular domain of CD20 is insoluble. Although the CD20 molecule derived from a cell lysate or as a gene recombinant protein can be solubilized by using a surfactant or strong alkali, it is difficult to maintain the natural three-dimensional structure under such a treatment condition. Therefore, a CD20 positive B cell strain is used as an immunogen for obtaining antibodies. However, immunostimulating property thereof is weak, and it is not easy to obtain clones of mature antibody-producing cells.
As of 2005, rituximab, a mouse/human chimeric antibody, is the only anti-CD20 monoclonal antibody approved as a therapeutic agent. Since chimeric molecules with heterologous molecules have antigenicity, they are not generally preferred as therapeutic agents. However, anti-CD20 antibodies have a property of targeting and eliminating all B cells including normal cells, and therefore they are said to have substantially no antigenicity. However, examples have been reported in which a neutralizing antibody is induced during the treatment period, although they account for only several percents, and it would become more likely to be induced depending on the dose and dosing period. Therefore, development of a humanized antibody having a sequence closer to that of human or a human antibody is desired. Another disadvantage of chimeric antibodies is the short blood half-life, and β half-life is only 3 or 4 days. The effective rate of rituximab alone against recurrence of low malignancy NHL was a little lower than 50% in a clinical study in the United States, indicating that 50% or more patients do not respond or poorly respond to rituximab. The response rate in patients with moderate malignancy NHL is even lower, being only about 30% (Non-patent document 14). Therefore, it is necessary to investigate the factors and background of the different responses in patients, and development of an antibody having a superior effect is desired at the same time.    Non-patent document 1: Leukocyte Fact Book 2nd Edition, Academic Press    Non-patent document 2: Stashenko P et al., J. Immunol., 1980, 125: 1678-85    Non-patent document 3: Anderson K C et al., Blood, 1984, 63:    Non-patent document 4: Shan D et al., Blood, 1998, 91: 1644    Non-patent document 5: Flieger D et al., Cell Immunol., 2000, 204: 55-63    Non-patent document 6: Mathas S et al., Cancer Res., 2000, 60: 7170-6    Non-patent document 7: Cardarelli P M et al., Cancer Immunol. Immunother., 2002, 51: 15-24    Non-patent document 8: Pedersen I M et al., Blood, 2002, 99:    Non-patent document 9: Deans J P et al., Immunol., 2002, 107:    Non-patent document 10: Golay J T et al., J. Immunol., 1992, 149: 300-8    Non-patent document 11: Bourger I et al., Eur. J. Immunol., 1993, 23: 768-71    Non-patent document 12: White M W et al., J. Immunol., 1991, 146: 846-53    Non-patent document 13: Shan D et al., Cancer Immunol. Immunother., 2000, 48: 673-83    Non-patent document 14: Coiffier B et al., Blood, 1998, 92:    Non-patent document 15: Edward J C et al., Rheumatology (Oxford), 2001, 40: 205-11    Non-patent document 16: Zaja F et al., Heamatologica, 2002, 87: 189-95    Non-patent document 17: Perrotta S et al., Br. J. Haematol., 2002, 116: 465-7    Non-patent document 18: Polyak M J et al., Blood, 2002, 99: 3256-62